Project Summary/Abstract Transcription factors drive dynamic, cell-type specific, gene expression to define cell fate and functionality. Current optical microscopy technologies now enable direct visualization of transcription factors in live cells but cannot modulate transcription factor activity, which is required for delineating the contribution of genotypic modulation and phenotypic response. The emerging non-neuronal optogenetics provides a new strategy to regulate gene transcription, either by recruiting a transcription activation domain to a specific promoter or by photo-uncaging a sequestered transcription factor. Unlike native transcription factors, which regulates hundreds and thousands of target genes, the current optogenetic strategy only works for single- or a few gene targets and could suffer from high basal activity in the dark. Controlling multiplexed gene transcription with a larger library of transcription factors, thus, calls for an alternative strategy that empowers new modalities of optical control of gene transcription. The goal of this project is to fill this gap by developing a strategy based on the controlled rescue of protein degradation. In this strategy, base-level protein activities are suppressed by constant protein degradation until light triggers a burst of protein production. This strategy does not depend on the activation mechanism of the protein of interests and will significantly enhance the capacity of non-neuronal optogenetics. In this project, we present a plan within a four-year budget period to develop and validate the control native transcription factors. We will demonstrate blue-light-controlled T cell factor (TCF) downstream of the well- established Wnt signaling pathway (Aim 1) and develop an orthogonal optogenetic system to regulate the Notch intracellular domain (NICD)-mediate transcription with red light (Aim 2). Using our recently developed spatial light modulator, we will achieve precise multiplexing transcription control in space and time and ultimately achieve controlling the native transcription factors at the single-cell level (Aim 3). Our recent success in developing optogenetic tools for mammalian cells and Xenopus embryos well positions the applicant to carry out the proposed project. Results of this project will provide valuable assets to researchers who are interested in dissecting the spatial and temporal regulation of signal transduction during early embryonic development.